Methoprene-tolerant(Met) Mediated Signaling Pathways And Their Funtions In Leptinotarsa Decemlineata Larvae

The Colorado potato beetle Leptinotarsa decemlineata is the most serious pest of potato.It is an important invasive and quarantine pest.During development,L.decemlineata passes through four distinct phases,eggs,larvae,pupae and adults,under regulation of several hormones.Understanding of molecular modes to regulate the metamorphosis may faciligate the development of more effectively management strategies for L.decemlineata larvae.In the present study,we focused on the clarification of Met mediated JH signaling pathway.The results were as follows.1.Identification of the major components involved in JH signalingBased on L.decemlineata genome and transcriptome data,along with RT-PCR and RACE,the major componets involved in JH signaling cascade were identified.Among them were allatostatins LdAS-C and LdAS-B,Methoprene tolerant(LdMet),and Broad Complex(LdBrC).LdAS-C contained a C-type typical PVSCF sequence and LdAS-B contained B-type typical W(X6)W sequence.LdMet encoded a protein with a bHLH domain,two PAS domains,PAS-A and PAS-B,and a PAC domain.Five alternative splices of LdBrC gene(LdBrC-Z1,LdBrC-Z2,LdBrC-Z3,LdBrC-Z4,and LdBrC-Z6)were found.These alternative splices shaded a common BTB domain,but they differed with isoform specific C2H2 zinc finger domain.Accordingly,the JH signaling cascade was constructed.2.Confirmation of JH signaling cascadeThe temporal expression profiles revealed that the transcript levels of LdMet,LdKr-h1,LdHairy and LdBrC were parallel with JH titers.Five alternative splices of LdBrC gene(LdBrC-Zl,LdBrC-Z2,LdBrC-Z3,LdBrC-Z4,and LdBrC-Z6)were expressed specifically in fourth-instar larvae and prepupae.LdBrC-Z1 was highly expressed in prepupae;LdBrC-Z2 was abundantly expressed in the fourth-instar larvae 36 hours after ecdysis and in the day 3 prepupae;LdBrC-Z3 was highly expressed throughout all the fourth-instar stage;LdBrC-Z4 was abundantly expressed in the fourth-instar larvae 36 hours after ecdysis;LdBrC-Z6 was highly expressed in the day 3 prepupae.Ingestion of JH by the larvae stimulated the expression of LdMet but inhibited the transcription of LdBrC.RNA interference-aided knockdown of LdMet downregulated the expression levels of LdKr-h1 and LdHairy.Conversely,feeding dsLdMet and dsLdKr-hl significantly increased the transcript level of LdBrC.It seems that JH signaling represses LdBrC.3.Allatostatin LdAS-C inhibited JH synthesisLdAS-C and LdAS-B were both expressed in eggs,larvae,pupae and adults;LdAS-C was highly expressed during larval molts,in parallel with JH peaks,while LdAS-B was highly expressed in adults.LdAS-C was highly expressed in BR-CC-CA coplex and moderately expressed in midgut;LdAS-B was highly expressed in BR-CC-CA complex and moderately expressed in midgut and rectum.Ingestion of dsRNAs successfully knocked down target genes,ingestion of LdAS-C for 3 and 6 days upregulated expressions of LdJHAMT,LdMet and LdKr-hl,while ingestion of LdAS-B had no effects on their expressions compared with control larvae.Moreover,after one,two and three days’ingestion of dsLdAS-C,the relative JH levels in the hemolymph of treated larvae were 2.5,4.2 and 1.9 fold higher than those in control beetles.Furthermore,ingestion of LdAS-C repressed larval growth and delayed developments.In addition,knockdown of LdMet repressed the expression of LdAS-C.These results indicate that LdAS-C is the functional allatostatin;it is regulated by Met-mediated JH signal and forms a negative feedback to tune JH titer.4.LdBrC determined pupationIngestion of 20E induced the expression of these isoforms and knockdown of LdEcR reduced their mRNA levels.RNA infterference-aided knockdown of all these isoforms increased larval mortality,reduced larval weights and completely impaired pupation.RNAi of each splice decreased larval weights,but had different effects on pupation.Larvae fed on dsLdBrC-Z1 failed to pupate.Larvae having ingested dsLdBrC-Z2 could initiate larva-pupa molting but was not unable to finish the pupation.A portion of larvae having consumed dsLdBrC-Z6 could not initiate larva-pupa molting,with prematurely developed wings.In contrast,larvae fed on LdBrC-Z3 or LdBrC-Z4 pupated successfully.These results demonstrated LdBrC is essential for pupation and five alternative splices function differently.5.LdMet mediated JH-PTTH-20E signal affected the larval sizeIngestion of dsRNA originating from LdMet successfully knocked down target gene.In LdMet RNAi larvae,two JH response genes,LdKr-hl and LdHairy,were significantly downregulated and did not respond to dietary introcuced JH.Larvae fed with JH had a larger critical weight,delayed the duration from ecdysis to the attainment of critical weight,and increased larval and pupal weights.In contrast,the LdMet RNAi larvae had a smaller critical weight,a shorter duration from ecdysis to the attainment of critical weight,and decreased larval and pupal weights.Larvae fed on the mixture of both JH and dsLdMet performed similar to those fed on dsLdMet.These results suggested that JH regulated critical weight,growth period and final weight.Ingestion of JH repressed mRNA levels of genes involved in PTTH signaling,20E synthesis and 20E signaling.Conversely,ingestion of dsLdMet enhanced these mRNA levels;ingestion of both JH and dsLdMet had same results with dsLdMet.Therefore,JH,acting through its receptor Met,inhibits PTTH production and release before the attainment of critical weight.Once the critical weight is reached,JH production and release are averted,and the hemolymph JH is removed.The elimination of JH allows the brain to release PTTH.PTTH subsequently stimulates ecdysteroid biosynthesis and release to start larval-pupal transition in L decemlineata.3.JH-PTTH cassettes regulated light preference switch in wandering larvaeWandering L.decemlineata larvae stop feeding,leave plants and pupate in soil.The light preference switch of wandering larvae was studied in this chapter.At middle wandering phase,L.decemlineata larvae switched their phototactic behavior,from photophilic at foraging period to photophobic.Ingestion of JH,dsLdAS-C,dsLdJHEH1 or dsLdJHDK to enhance JH signaling significantly delayed light preference switch.Conversely,ingestion of dsLdJHAMT or dsLdMet to disturb JH signaling obviously accelerated this switch.RNAi of LdPTTH or LdTorso weakened photophobism of wandering larvae.The resultand larvae pupated on soil or in shallow soil,in a 20E-independent pattern.Furthermore,a combination of depletion of LdPTTH/LdTorso and disturbance of JH signal resulted in similar phenotypes to the LdPTTH or LdTorso RNAi larvae.The mRNA levels of LdTrpA,a transient receptor potential(TRP)cation channel gene,were reduced in larvae fed on dsLdPTTH or dsLdTorso.Finally,larvae fed with dsLdTrpA1 or a combination of dsLdTrpA1 and dsLdPTTH or dsLdTorso showed similar photophobism and pupation behaviors to the LdPTTH or LdTorso RNAi larvae.These results demonstrated JH-PTTH cascade regulates both metamorphosis and light preference switch.